This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.
Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by a genetic defect in the SMN1 gene, which encodes survival motor neuron (SMN) protein, which is widely expressed in eukaryotic cells. SMN protein is critical to the function of the nerves that control muscles (the motor neurons). Diminished abundance of the protein results in loss of function of neuronal cells in the anterior horn of the spinal cord and subsequent system-wide muscle atrophy. The condition is debilitating and often fatal.
A second gene also has a role in producing SMN protein. This is the survival motor neuron 2 gene (SMN2), often called the SMA “back-up gene.” However, most of the SMN protein produced by SMN2 lacks a key building block that is normally produced by SMN1. This means that SMN2 cannot fully make up for the mutated SMN1 gene.
Thus, spinal muscular atrophy is caused by loss or mutation of the SMN1 gene and retention of the SMN2 gene [Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M. et al. (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell, 80, 155-165; Burghes, A. H. and Beattie, C. E. (2009) Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci, 10, 597-609]. As has been described, the SMN2 gene differs from the SMN1 gene by a single nucleotide, which disrupts a splice modulator in exon 7 in SMN2 [Lorson, C. L., Hahnen, E., Androphy, E. J. and Wirth, B. (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA, 96, 6307-6311; Monani, U. R., Lorson, C. L., Parsons, D. W., Prior, T. W., Androphy, E. J., Burghes, A. H. and McPherson, J. D. (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet, 8, 1177-1183; Cartegni, L. and Krainer, A. R. (2002) Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet, 30, 377-384; Kashima, T. and Manley, J. L. (2003) A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet, 34, 460-463]. The disruption of this splice modulator results in the majority of the transcript from SMN2 lacking exon 7 and, in turn, the SMN protein lacking exon 7 does not self-associate readily and is rapidly degraded [Gennarelli, M., Lucarelli, M., Capon, F., Pizzuti, A., Merlini, L., Angelini, C., Novelli, G. and Dallapiccola, B. (1995) Survival motor neuron gene transcript analysis in muscles from spinal muscular atrophy patients. Biochem Biophys Res Commun, 213, 342-348; Parsons, D. W., McAndrew, P. E., Monani, U. R., Mendell, J. R., Burghes, A. H. and Prior, T. W. (1996) An 11 base pair duplication in exon 6 of the SMN gene produces a type I spinal muscular atrophy (SMA) phenotype: further evidence for SMN as the primary SMA-determining gene. Hum Mol Genet, 5, 1727-1732; Lefebvre, S., Burlet, P., Liu, Q., Bertrandy, S., Clermont, O., Munnich, A., Dreyfuss, G. and Melki, J. (1997) Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet, 16, 265-269; Coovert, D. D., Le, T. T., McAndrew, P. E., Strasswimmer, J., Crawford, T. O., Mendell, J. R., Coulson, S. E., Androphy, E. J., Prior, T. W. and Burghes, A. H. (1997) The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet, 6, 1205-1214; Lorson, C. L. and Androphy, E. J. (2000) An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum Mol Genet, 9, 259-265; Lorson, C. L., Strasswimmer, J., Yao, J. M., Baleja, J. D., Hahnen, E., Wirth, B., Le, T., Burghes, A. H. and Androphy, E. J. (1998) SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat Genet, 19, 63-66; Burnett, B. G., Munoz, E., Tandon, A., Kwon, D. Y., Sumner, C. J. and Fischbeck, K. H. (2009) Regulation of SMN protein stability. Mol Cell Biol, 29, 1107-1115]. This results in less SMN protein and SMA.
More specifically, human chromosome 5 contains two nearly identical genes at location 5q13: a telomeric copy SMN1 and a centromeric copy SMN2. In healthy individuals, the SMN1 gene codes the survival of motor neuron protein (SMN) which, as its name says, plays a crucial role in survival of motor neurons. The SMN2 gene, on the other hand—due to a variation in a single nucleotide (840.C→T)—undergoes alternative splicing at the junction of intron 6 to exon 8, with only 10-20% of SMN2 transcripts coding a fully functional survival of motor neuron protein (SMN-fl) and 80-90% of transcripts resulting in a truncated protein compound (SMNΔ7) which is rapidly degraded in the cell.
In individuals affected by SMA, the SMN1 gene is mutated in such a way that it is unable to correctly code the SMN protein—due to either a deletion occurring at exon 7 or to other point mutations (frequently resulting in the functional conversion of the SMN1 sequence into SMN2). All patients, however, retain at least one copy of the SMN2 gene (with most having 2-4 of them) which still codes small amounts of SMN protein—around 10-20% of the normal level—allowing some neurons to survive. In the long run, however, reduced availability of the SMN protein results in gradual death of motor neuron cells in the anterior horn of spinal cord and the brain. Muscles that depend on these motor neurons for neural input now have decreased innervation (also called denervation), and therefore have decreased input from the central nervous system (CNS). Denervated skeletal muscle is more difficult for the body to control. Decreased impulse transmission through the motor neurons leads to decreased contractile activity of the denervated muscle. Consequently, denervated muscles undergo progressive atrophy.
Spinal muscular atrophy manifests in various degrees of severity, which all have in common progressive muscle wasting and mobility impairment. Proximal muscles and lung muscles are affected first. Other body systems may be affected as well, particularly in early-onset forms. SMA is the most common genetic cause of infant death.
The copy number of SMN2 inversely correlates with patient severity and increased full-length SMN from an SMN2 gene also correlates with a milder phenotype [McAndrew, P. E., Parsons, D. W., Simard, L. R., Rochette, C., Ray, P. N., Mendell, J. R., Prior, T. W. and Burghes, A. H. (1997) Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet, 60, 1411-1422; Mailman, M. D., Heinz, J. W., Papp, A. C., Snyder, P. J., Sedra, M. S., Wirth, B., Burghes, A. H. and Prior, T. W. (2002) Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med, 4, 20-26; Feldkotter, M., Schwarzer, V., Wirth, R., Wienker, T. F. and Wirth, B. (2002) Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet, 70, 358-368; Prior, T. W., Krainer, A. R., Hua, Y., Swoboda, K. J., Snyder, P. C., Bridgeman, S. J., Burghes, A. H. and Kissel, J. T. (2009) A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet, 85, 408-413]. The severity of SMA symptoms is broadly related to how well the remaining SMN2 genes can make up for the loss of SMN1. This is partly related to the number of SMN2 gene copies present on the chromosome. Patients with SMA can have between 1 and 4 (or more) copies, with the greater the number of SMN2 copies, the milder the disease severity. Thus, most SMA type I patients have one or two SMN2 copies; SMA II and III patients usually have at least three SMN2 copies; and SMA IV patients normally have at least four of them.
Generally, patients tend to deteriorate over time, but prognosis varies with the SMA type and disease progress which is most rapid in SMN type I.
The majority of children diagnosed with SMA type 0/I do not reach the age of 2 without respiratory intervention—recurrent respiratory problems being the primary cause of morbidity. With proper care, milder SMA type II and III cases live into adulthood.
In SMA type II, the course of the disease is stable or slowly progressing and life expectancy is reduced compared to the healthy population. Death before the age of 20 is frequent, although many patients live to become parents and grandparents.
SMA type III has near-normal life expectancy if standards of care are followed. Adult-onset SMA usually means only mobility impairment and does not affect life expectancy.
Thus increasing, enhancing, and/or restoring SMN levels should have a beneficial impact on the SMA phenotype in a subject (e.g., humans).
There is no known cure for spinal muscular atrophy. And so, care largely involves treating the symptoms of SMA (e.g., palliative care). However, since the underlying genetic mechanism of SMA was described in 1990, several therapeutic approaches have been proposed and investigated. Since a vast number of in vitro and animal modelling studies suggest that restoration of SMN levels reverts SMA symptoms, the majority of emerging therapies focus on increasing the availability of SMN protein to motor neurons. Such therapeutic pathways include gene replacement and SMN2 gene conversion.
Gene therapy aims at correcting the SMN1 gene function through inserting specially crafted nucleotide sequences with the help of a viral vector. In the context of SMA, it is currently being researched using the scAAV9 viral vector at the Ohio State University and Nationwide Children's Hospital, USA, and the University of Sheffield, United Kingdom, as well as by Genzyme Corporation, USA, and Généthon, France. In one study this method has resulted in the greatest survival increase achieved to-date in a SMNΔ7 mouse model (median survival of 400 days in treated mice as opposed to 15 days in untreated mice) [Foust, Nature Biotech. 28, 271-274 (2010)]. Safety and pharmacokinetics of scAAV9 viral vector has been tested in non-human primates [Meyer, Molecul Ther (31 Oct. 2014) I doi:10. 1038/mt.2014.210]. An AAV9 viral vector is in clinical trials for Hemophilia B. scAAV9-SMN is in clinical trials currently at NCH Clinical Trials.gov Identifier: NCT02122952.
SMN2 gene conversion, also known as ‘SMN2’ alternative splicing modulation, essentially aims at converting the “backup” SMN2 gene (which normally produces only a fraction of needed SMN protein) into a fully functional SMN1 gene so as it is able to code for high quantities of full-length SMN protein.
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA.
This synthesized nucleic acid is termed an “anti-sense” oligonucleotide because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA “5′-AAGGUC-3′” would be blocked by the anti-sense mRNA segment “3′-UUCCAG-5′”).
However, to date, no truly effective therapy has been developed (i.e., one which produces enough full length SMN protein to eliminate the effects of SMA, cure SMA, and/or result in extended survival times and rates.